Project Summary/Abstract Pain is a silent epidemic in the United States, with an estimated 76.2 million Americans living with chronic pain caused by disease, disorder or accident. Importantly, many serious diseases such as cancer, heart disease, AIDS and arthritis are often associated with unrelieved pain. Pain sensation (i.e. nociception) is initiated when the peripheral terminals of nociceptors, a subgroup of sensory neurons, are activated by noxious chemical, mechanical or thermal stimuli. However, nociception is a complex process that involves the activation of a myriad of molecules and proteins, leading to crosstalk among numerous signaling pathways. The identification and characterization of the Transient Receptor Potential (TRP) family of receptors has provided immense insight into understanding the complex processes involved in pain transmission. The first TRP member identified, TRPV1 (Transient Receptor Potential Vanilloid 1), has been the most extensively studied with regards to pain. Recently, several studies have demonstrated that TRPV1 association with scaffolding proteins plays a critical role in the regulation of the receptor. Interestingly, Beta-arrestins a family of cytosolic proteins initially identified for their central role in G-protein coupled receptor (GPCR) desensitization are now attributed with novel roles as scaffolding proteins, which modulate the strength and duration in signaling of a variety of receptors. Preliminary data from our laboratory demonstrates Beta-arrestin association with TRPV1 in primary cultures of trigeminal (TG) sensory neurons. Moreover, overexpression of Beta-arrestin and TRPV1 in a heterologous cell culture model leads to increased receptor desensitization following repeat applications of the selective agonist capsaicin compared to cells transfected alone with TRPV1. To elucidate the contribution of Beta-arrestin in TRPV1 desensitization, additional experiments were conducted which confirmed association of the phosphodiesterase PDE4D with Beta-arrestin in TG neurons. Further investigation to reveal the physiological relevance of Beta-arrestin association with the TRPV1 receptor may provide greater insight into the treatment and management of a variety of pain disorders. The central hypothesis of our proposal is that Beta-arrestin negatively regulates the TRPV1 receptor. Our specific aims are to: 1) Investigate the role of TRPV1 phosphorylation in Beta-arrestin association. 2) Determine the effect of Beta-arrestin knockdown on TRPV1 activity. 3) Investigate Beta-arrestin-scaffolded PDE4D in the regulation of TRPV1.